JPS5854129B2 - Lymphocyte separation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for separating lymphocytes from blood, body fluids, or a blood cell suspension obtained by processing these in a short time with high purity and high yield, and with simple operations. .

To explain in more detail, the blood cell suspension is first passed through a filter that captures granulocytes and monocytes, and after separating the granulocytes and monocytes from other components, the blood cell suspension containing few granulocytes and monocytes is filtered into white blood cells. Lymphocytes are collected by passing the blood cells through a filter that captures them, separating leukocytes, which are mostly lymphocytes, from other components in the blood cell suspension, and then collecting the lymphocytes captured by the filter. This relates to a method of separating.

White blood cells generally consist mostly of granulocytes, monocytes, and lymphocytes, and therefore, when granulocytes and monocytes are removed from a leukocyte suspension, a lymphocyte suspension is obtained.

In recent years, with advances in immunology, lymphocytes have been extracted from the blood in pure form and this lymphocyte suspension has been used to conduct immunological research such as histocompatibility antigens and cellular immunity, as well as research on various diseases. It has come to be used in immunotherapy, and these lymphocytes have been separated into subpopulations such as T cells and B cells, and helper T cells and suppressor T cells have been used.
Attempts to separate cells into subsets are underway in hospitals and
It is being considered by research institutions.

However, there has been no suitable method for obtaining a relatively large amount of lymphocyte suspension used for these purposes from blood or the like, with simple operations, and with good purity and yield.

Conventional lymphocyte separation methods that can be used for this purpose include density gradient centrifugation such as the Ficoll-Conray method, cell electrophoresis, and adding a hemagglutinating agent to blood to precipitate and remove red blood cells. , the obtained leukocyte suspension,
There is a method to obtain a lymphocyte suspension by bringing granulocytes and monocytes into contact with a substance that adheres, adheres, or adsorbs them, and then removing them. It is difficult to obtain lymphocytes in a timely manner, and problems may arise when transfusing the obtained lymphocyte suspension into the human body.

Therefore, the present inventors have developed a method to extract blood from a large amount of blood using a simple procedure.
As a result of intensive research into a method that can separate lymphocytes with high purity and high yield in a short period of time, and which does not cause any problems even when the lymphocyte suspension is transfused into the human body, we have developed a granulocyte separation filter that captures granulocytes and monocytes. They discovered that this could be achieved by combining a leukocyte separation filter that captures leukocytes, and completed the main goal.

That is, in the present invention, blood, body fluid, or a blood cell suspension obtained by processing the same is passed through a filter that captures granulocytes and a single row, and after separating granulocytes, monocytes, and pore components, the granules are separated. A lymphatic system characterized by passing a suspension of positive cells with few monocytes and leukocytes through a filter that captures white blood cells to separate marimba cells and other components, and then collecting the lymphocytes captured by a filter. This is a method to obtain a lymphocyte suspension containing few blood cell components other than cells.

In the present invention, blood, body fluid, or a blood cell suspension obtained by processing these refers to blood or body fluid, for example, ascites fluid or bone marrow fluid itself, and the treatment of these fluids, such as dextran or hydroxyl. Blood cell suspension obtained by adding a blood agglutinating agent such as ethyl starch, blood cell suspension obtained by centrifugation such as density gradient centrifugation, and blood cell suspension obtained by cell electrophoresis. Power is called.

The method according to the present invention uses both a granulocyte separation filter and a leukocyte separation filter C to obtain lymphocytes from these blood cell suspensions.

It has been well known that granulocytes and monocytes adhere or adhere to various materials, such as cotton, polyester fibers, nylon fibers, and silicon-treated glass wool, and that lymphocytes also weakly adhere to these materials. It is a phenomenon.

However, simply contacting blood with these substances only removes granulocytes, Korean cells, and a very small amount of lymphocytes in the blood, while a large amount of red blood cells are recovered together with the majority of lymphocytes. In order to obtain only lymphocytes, it is necessary to remove the red blood cells.

Currently, this is accomplished by density gradient centrifugation or by contacting the red blood cells with a hypotonic solution to destroy them.

However, these operations are time-consuming and difficult to process large amounts of blood.

Therefore, the present inventors investigated various ways to obtain lymphocytes by combining a granulocyte separation filter, and found that by combining a leukocyte separation filter and a granulocyte separation filter, lymphocytes can be obtained from blood in a short time with simple operations. We discovered that spheres can be separated with high purity and high yield.

A white blood cell separation filter can capture white blood cells from blood, and by washing the inside of the filter with a physiological solution such as physiological saline that does not collect white blood cells, most components other than white blood cells can be removed. Any material that can easily collect leukocytes may be used.

One example of this is to fill a column with fibrous material and utilize the adhesion of white blood cells to the fibrous material or the trapping of white blood cells by trapping them in the spaces created by the fibrous material.

These are different from conventionally known granulocyte separation filters in that they take into consideration the above points so that lymphocytes can be captured, but they use fibrous substances with an average diameter of 10 μ or less. It is preferable to use a filter because it can increase the surface area to which leukocytes can adhere, and can also reduce the space created by fibrous substances.

In this case, the fibrous material is one whose length is much longer than its average diameter.

Also, the average diameter means that the weight of the object is Xl and the length is y.
CIrt, where the density is ρP/i, is defined by D=2/Rhoji.

The captured white blood cells can be collected by, for example, adding an adhesion inhibitor or flowing the liquid while applying physical force, if the white blood cells have some kind of adhesion to the substance inside the filter. If the liquid is reaching the space created by the substance inside the filter, it can be recovered by changing the size of the space or by applying physical force from the outside to cause the liquid to flow.

In addition, the granulocyte separation filter does not allow lymphocytes to get stuck in the space created by the substances inside the filter, nor does it non-specifically adhere to lymphocytes, allowing it to capture granulocytes and monocytes well. It is something.

An example of this is commercially available filters whose columns are filled with polyester fibers, nylon fibers, etc.

In the present invention, lymphocytes can be separated by simple operations using these two filters.

FIG. 1 is a process explanatory diagram showing one example of the lymphocyte separation method according to the present invention.

In the figure, 1 is an inlet for liquid entry, 2 is an outlet for liquid exit, 3 is a granulocyte separation filter, 4 is a leukocyte separation filter, and 5 is a part that connects these.

The blood cell suspension entering from 1 passes through a granulocyte separation filter 3 and a leukocyte separation filter 4 and exits from an outlet 2.

During this time, some of the granulocytes, monocytes, and red blood cells in the blood cell suspension are captured by the granulocyte separation filter 3, and the leukocyte separation filter 4 contains white blood cells, mostly composed of lymphocytes, and other blood cells. Some of the components are captured.

Next, a physiological solution that does not collect granulocytes, monocytes, or lymphocytes is sent through the inlet 1, and components other than white blood cells contained in the granulocyte separation filter 3 and the leukocyte separation filter 4 are flushed out through the outlet 2.

Thereafter, a liquid for collecting lymphocytes is sent from the inlet 1, and the lymphocytes captured in the leukocyte separation filter 4 are taken out from the outlet 2.

FIG. 2 is also a process explanatory diagram showing one example of the lymphocyte separation method according to the present invention.

In the figure, 1 is the same as in Figure 1, the inlet where the liquid enters the main part where lymphocytes are separated, 2 is the outlet, 3 is the granulocyte separation filter, and 4
5 is a leukocyte separation filter, 5 is a connection, 6 is a pump for sending liquid, 7 is an inlet for liquids other than 1, and 8 is a cock for sending the liquid to an appropriate location.

The blood cell suspension passes through the inlet 1, passes through the granulocyte separation filter 3 and the leukocyte separation filter 4, and exits from the outlet 2. During this time, the granulocytes and monocytes are captured by the granulocyte separation filter 3, and then passed through the leukocyte separation filter 4. white blood cells, mostly lymphocytes, are captured.

However, in addition to granulocytes and monocytes, the granulocyte separation filter 3 traps lymphocytes and some other blood cells, so when you want to collect these lymphocytes as well, be careful not to elute the granulocytes and monocytes. Preferably, the physiological solution is passed through the inlet 1 into the granulocyte separation filter 3 and then into the leukocyte separation filter 4.

Next, a washing liquid that does not collect white blood cells or lymphocytes is flowed through the leukocyte separation filter 4 from the input lower to flush out red blood cells and platelets in the filter.

Thereafter, a liquid for collecting white blood cells or lymphocytes is sent from the inlet lower, and a lymphocyte suspension is obtained from the outlet 2.

Note that these figures only show a part of the method according to the present invention; for example, in Fig. 1, two filters are connected, but in this case, each part is separated and the granulocyte separation filter 3 is used. Take out the blood cell suspension from which granulocytes and monocytes have been removed, and pass this liquid through a leukocyte separation filter 4.
Lymphocytes can also be separated by placing them in a

It is also possible that no cleaning liquid is used.

The location of the cock and the connections of the pipes are only shown as examples.

When lymphocytes are directly isolated from a donor, in addition to this method, by connecting a connection part for taking out venous blood and a return part to the donor, a large amount of blood cells other than lymphocytes can be returned to the donor. lymphocytes can be obtained.

As an example of the lymphocyte separation method according to the present invention, the cases shown in FIGS. 1 and 2 have been described, but gravity may be used in addition to a pump as the force for sending the liquid.

The lymphocyte suspension obtained in this way has less contamination with red blood cells, platelets, granulocytes, and monocytes, and the operation for obtaining it is also very simple.

In addition, the obtained lymphocytes retain their ability to become young by various mitogens, such as phytohemagglutinin and Bork Weed mitogen, and T cells,
Depending on the conditions of use of the filter, the ratio of B cells may be the same as the original blood or may be different.

As described above, according to the present invention, lymphocytes can be separated from blood cell suspensions such as blood with simple operations and aseptically with high purity and yield, and lymphocyte suspensions can be used in various medical facilities. This allows for easy research and transfusion of lymphocyte components.

Example 1 Using a granulocyte separation filter packed with 1.5 denier polyester fibers with a length of 4CrfL to 7cIrL and a leukocyte separation filter filled with 0.7 denier polyacrylonitrile fibers with a length of 4cmjJ x 7, By the method shown in FIG. 2, lymphocytes were separated from 100 ml of blood containing 1000 units of heparin from a healthy person.

First, pump 100ml of blood to 3ml/wtiy using pump 6.
The mixture was passed through a granulocyte separation filter 3 at a flow rate of t(7), and then directly passed through a leukocyte separation filter 4.

Next, in order to introduce the lymphocytes remaining in the granulocyte separation filter 3 into the leukocyte separation filter 4, physiological saline is poured into the granulocyte separation filter 3 for 30 m at a flow rate of 31 rLl/-.
1, and this liquid was directly passed through the leukocyte separation filter 4.

Next, in order to remove blood cell components other than lymphocytes captured in the leukocyte separation filter 4, 2001
rLl of physiological saline was flowed at 10 ml/mm and exited from outlet 2.

After this, in order to collect the lymphocytes captured in the leukocyte separation filter 4, a physiological solution containing plasma is added to the 100 m
l, 5rnl! The mixture was passed through the filter at a flow rate of /mltt while applying physical force, and 100TLl of the lymphocyte suspension was collected.

This fluid contains 53% of lymphocytes, granulocytes,
It contained 6% monocytes, 0.3% red blood cells, and 3% platelets.

Example 2 A granulocyte separation filter packed with 1.5 denier polyester fibers with a length of 4α to 7 crrL and a leukocyte separation filter packed with 0.7 denier polyamide fibers with a length of 4 crrL to 7 crrL were used. By the method shown in Example 1, a lymphocyte suspension was obtained from 1 ooml of blood of a healthy person.

ml was obtained.

This fluid contains 48% of lymphocytes, granulocytes,
It contained 5% monocytes, 0.3% red blood cells, and 4% platelets.

Example 3 - Granulocyte separation filter packed with polyester fibers of 1.5 denier and length 4 (1771 to 7 CrfL) and
Using a white blood cell separation filter filled with polyacrylonitrile synthetic fibers with a length of 7 denier and 4 crrL to 7 cm, 10 pieces of blood from a healthy person were collected by the method shown in Figure 2.
Lymphocyte suspension 1 was prepared in the same manner as in Example 1 from a solution containing 5% of the original red blood cells and 70% of the original lymphocytes, which was obtained by adding a dextran solution with a molecular weight of 200,000 to 0ml.
00ml was obtained.

This fluid contains 36% of the original blood lymphocytes, granulocytes,
It contained 3% monocytes, 0.02% red blood cells, and 0.3% platelets.

[Brief explanation of drawings]

FIG. 1 and FIG. 2 are process explanatory diagrams showing an example of the lymphocyte separation method according to the present invention. 1... Inlet of blood cell suspension, 2... Outlet of blood cell suspension, 3... Granulocyte separation filter,
4... Leukocyte separation filter 5...
Connection part, 6...Pump, 7...Inlet,
8...Cook.

Claims (1)

[Claims] 1 Blood, body fluids, or a blood cell suspension obtained by processing these are passed through a filter that captures granulocytes and hemispheres to separate granulocytes and monocytes from other components. The suspension is passed through a filter that captures white blood cells, the lymphocytes and other components are separated, and the lymphocytes captured by this filter are then collected. How to obtain a lymphocyte suspension.